The order Homoptera, often regarded as a separate suborder of the order Hemiptera, includes those insects known as plant bugs. These insects have piercing and sucking mouth-parts and feed upon sap. They include the aphids [family Aphididae], white flies [Aleyrodidae], planthoppers [Delphacidae], leafhoppers [Cicadellidae], jumping plant lice [Psyllidae] woolly aphids [Pemphigidae], mealy bugs [Pseudococcidae], and scales [Coccidae, Diaspididae, Asterolecaniidae and Margarodidae]. Many species are serious pests of agricultural and horticultural crops and of ornamental plants, including, for example, pea aphid, black bean aphid, cotton aphid, green apple aphid, glasshouse-potato aphid, leaf-curling plum aphid, banana aphid, cabbage aphid, turnip aphid, peach-potato aphid, corn leaf aphid, wheat aphid, brassica whitefly, tobacco whitefly, glasshouse whitefly, citrus blackfly, small brown planthopper, rice brown planthopper, sugarcane planthopper, white-backed planthopper, green rice leafhopper, beet leafhopper, cotton jassid, zig-zag winged rice leafhopper, apple sucker, pear sucker, woolly apple aphid, lettuce root woolly aphid, grape phylloxera, long-tailed mealybug, pineapple mealybug, striped mealybug, pink sugarcane mealybug, cottony cushion scale, olive scale, mussel scale, San Jose scale, California red scale, Florida red scale and coconut scale.
Crop damage as a result of feeding by these insects occurs in a number of ways. Extraction of sap deprives the plant of nutrients and water leading to loss of vigour and wilting. Phytotoxic substances present in the saliva of some species, and mechanical blockage of the phloem by feeding may result in distortion and necrosis of foliage [as in `hopper-burn`] and in blindness or shrunken kernels in grain crops. Injury, caused by insertion of the mouthparts leaves lesions through which plant pathogens may enter. Production of copious `honeydew` may allow sooty moulds to develop or its stickiness may interfere with the harvesting of cereals and cotton. Some of the most serious damage caused by these pests is indirect, due to their role as vectors of plant viruses. Examples of serious virus diseases spread by Homopterans include maize streak, beet curly-top, northern cereal mosaic, oat rosette, pear decline, tobacco mosaic, cauliflower mosaic, turnip mosaic, rice orange leaf, rice dwarf, rice yellow dwarf, rice transitory yellowing, rice grassy stunt, sugarcane Fiji disease, cassava mosaic, cotton leaf-curl, tobacco leaf-curl, sweet potato virus B, groundnut rosette, banana bunchy top, citrus tristeza, pineapple mealybug wilt and cocoa swollen shoot. Reduction in the Homopteran insect populations would be useful in limiting the spread of these and other viral diseases in crops. This invention addresses the problem of control of these sucking insect pests.
Since the late 1940s, methods to control these pests have centred on the exogenous application of synthetic organochemicals. Because of their feeding habits, effective insecticides must act on contact or be systemic within the plant. Insecticides of the chlorinated hydrocarbon, substituted phenol, organophosphate, carbamate and pyrethrin classes have been used, but this method of plant protection is encountering increasing problems known to those versed in the art. The problem of the development of pest insect resistance to pesticides is particularly acute amongst Homopterans, where the typically short generation time allows the emergence of resistant biotypes very rapidly. For example, the brown planthopper of rice can apparently develop a new biotype in only about 18 months.
Biological control of pest insects has been favoured as an alternative strategy. Such an approach exploits the natural viral, bacterial or fungal pathogens or the natural invertebrate or vertebrate predators of the target pest to limit its population. Examples include granulosis and polyhedrosis viruses effective against specific caterpillar and sawfly larvae [eg. Heliothis PHV] and Bacillus thuringiensis kurstaki, effective against certain caterpillars. Pathogenic/parasitic fungi have been identified in a wide range of insect classes, including Homopterans; Venicilium lecanii has proved useful in the control of aphids and whitefly in glasshouses, though not in the field. Free living and parasitic predators of pests have been used with some success, particularly in stable ecosystems such as glasshouses and orchards, for example various Hymenopteran, Coleopteran and Acarinan insects have been used for the control of aphids and whitefly in glasshouses. The widespread introduction of biological control measures has, however, been limited by problems of large scale production, widespread application and lack of persistence of the control agents in the field.
A preferred solution is to use inherently insect resistant cultivars, varieties or lines as part of an integrated pest management programme. Production of such resistant lines, which may exhibit pest avoidance, non-preference, antibiosis or pest tolerance, is a major goal of many conventional plant breeding programmes for crop improvement. In those cases where a biochemical mechanism of resistance to Homopterans has been determined, it resides in altered levels of specific, non-protein secondary plant metabolites. This approach is limited by the extent of genetic variability for resistance which is available within the interbreeding germplasm. Often a source of resistance to a specific pest is not available. It is further limited by the long time scale required to produce a resistant cultivar imposed by the large number of backcrosses necessary to introgress the character into an agronomically acceptable genetic background. In response to the introduction of new insect resistant lines of plants, new insect biotypes arise to which the plants are not resistant. As with synthetic insecticides, development of resistance breaking biotypes is a particular problem with Homopterans. For example, many rice varieties resistant to the brown planthopper [BPH] have been developed at the International Rice Research Institute, Manila, Philippines [IRRI] which have given excellent control of this bug in the field for a time. However, resistance breaking biotypes have developed in some localities to the extent that local rice production is threatened. As fast as new BPH resistant varieties come out of the IRRI programme, new resistance breaking biotypes of the insect evolve. There is thus a continuous need for novel sources of BPH resistance genes to incorporate into the breeding programme.
Genetic engineering may make a useful contribution here since it allows genes encoding insecticidal proteins to be inserted into elite lines of crop plants in a single step irrespective of the natural source of the protein or its ability to cross-breed with the host. A number of cases are known to the art wherein a foreign gene is introduced into a transgenic plant which specifies the production of levels of protein which are toxic or antimetabolic to insects which feed on the plant by chewing the leaves, roots, stems or fruits.
European Patent Application 0142 924 [Agrigenetics Research Associates Ltd.] describes the production in transgenic plants of a B. thuringiensis delta endotoxin which is larvicidal to certain Lepidoptera. Similarly, EPA 0193 259 [Plant Genetic Systems NV] describes production of a Lepidopteran-toxic protein in transgenic plants expressing a truncated Bt toxin gene. Bt toxin genes useful for controlling Coleopteran insects on introduction into transgenic plants are described in EPA 0289 479 [Monsanto Co.] and EPA 0305 275 [Plant Genetic Systems NV]. EPA 0339 009 [Monsanto Co] describes the potentiation of effectiveness of a Lepidopteran specific Bt toxin by co-expressing trypsin inhibitor in transgenic plants.
EPA 0272 144 [Agricultural Genetics Co.] teaches that the expression of a gene encoding trypsin inhibitor from cowpeas in transgenic plants enhances resistance to both Lepidopteran and Coleopteran pests.
EPA 0337 750 [Plant Cell Research Institute Inc.] describes the introduction and expression of genes encoding arcelin, a storage protein of wild Phaseolus bean seeds, in transgenic plants and suggests that it might be useful in protection from Coleopteran pests.
EPA 0351 924 [Shell International Research Maatschappij BV] describes the introduction and expression of genes encoding the lectin from the garden pea in transgenic plants enhancing resistance to Lepidopteran larvae. PCT/GB91/01290 [Agricultural Genetics Co.] relates to the use of genes encoding lectins from the monocotyledonous Families Amaryllidaceae and Alliaceae for control of Lepidopterans and Coleopterans, without associated mammalian toxicity. EPA 0427 529 [Pioneer Hi-Bred International Inc.] describes the use of a variety of lectins to control Lepidopteran and Coleopteran pests.
Each of these proteins have to be ingested to exert their insecticidal effect, and their primary site of action appears to be the midgut [Hoffmann, C. et al. {19882} Proc. Natl. Acad. Sci. USA 85, 7844-; Gatehouse A. M. R. and Boulter, D. {1983} J. Sci. Food Agric. 34, 345-350; Gatehouse et al. {1984} J. Sci. Food Agric. 35, 373-380]. However, the digestive system in sap-sucking Homopterans is unusual amongst the Insecta in a number of important respects, for example they lack a crop, the midgut is not lined by a peritrophic membrane and hydrolytic digestive enzyrnes are absent [see eg. Terra, W. R. {1990} Annu. Rev. Entomol. 35, 181-200]. In view of the radically different mode of feeding, nature of food and necessary organisation of the gut between plant tissue chewing insects and sap-sucking insects, it would be very surprising if proteins which had insecticidal activity against one class of insects were to be effective against the other.